• Transcriptome profiling reveals Silibinin dose-dependent response network in non-small lung cancer cells.

      Kaipa, Jagan Mohan; Starkuviene, Vytaute; Erfle, Holger; Eils, Roland; Gladilin, Evgeny; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Peer J, 2020-12-16)
      Silibinin (SIL), a natural flavonolignan from the milk thistle (Silybum marianum), is known to exhibit remarkable hepatoprotective, antineoplastic and EMT inhibiting effects in different cancer cells by targeting multiple molecular targets and pathways. However, the predominant majority of previous studies investigated effects of this phytocompound in a one particular cell line. Here, we carry out a systematic analysis of dose-dependent viability response to SIL in five non-small cell lung cancer (NSCLC) lines that gradually differ with respect to their intrinsic EMT stage. By correlating gene expression profiles of NSCLC cell lines with the pattern of their SIL IC50 response, a group of cell cycle, survival and stress responsive genes, including some prominent targets of STAT3 (BIRC5, FOXM1, BRCA1), was identified. The relevancy of these computationally selected genes to SIL viability response of NSCLC cells was confirmed by the transient knockdown test. In contrast to other EMT-inhibiting compounds, no correlation between the SIL IC50 and the intrinsic EMT stage of NSCLC cells was observed. Our experimental results show that SIL viability response of differently constituted NSCLC cells is linked to a subnetwork of tightly interconnected genes whose transcriptomic pattern can be used as a benchmark for assessment of individual SIL sensitivity instead of the conventional EMT signature. Insights gained in this study pave the way for optimization of customized adjuvant therapy of malignancies using Silibinin.
    • Biogeography and Environmental Drivers of Abundance and Genotype Composition Across the West Bank: Relevance of a Genotype-Based Ecology for Understanding Occurrence.

      Zayed, Ashraf R; Butmeh, Suha; Pecellin, Marina; Salah, Alaa; Alalam, Hanna; Steinert, Michael; Höfle, Manfred G; Bitar, Dina M; Brettar, Ingrid; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-12-01)
      The West Bank can be considered as a high-risk area for Legionella prevalence in drinking water due to high ambient temperature, intermittent water supply, frequent pressure loss, and storage of drinking water in roof containers. To assess occurrence of Legionella species, especially L. pneumophila, in the drinking water of the West Bank, the drinking water distribution systems of eight hospitals were sampled over a period of 2.3 years covering the seasonal cycle and the major geographic regions. To gain insight into potential environmental drivers, a set of physico-chemical and microbiological parameters was recorded. Sampling included drinking water and biofilm analyzed by culture and PCR-based methods. Cultivation led to the isolation of 180 strains of L. pneumophila that were genotyped by Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). Surprisingly, the abundance of culturable L. pneumophila was low in drinking water of the sampling sites, with only three out of eight sites where Legionella was observed at all (range: 30-500 CFU/liter). By contrast, biofilm and PCR-based analyses showed a higher prevalence. Statistical analyses with physico-chemical parameters revealed a decrease of L. pneumophila abundance for water and biofilm with increasing magnesium concentrations (>30 mg/l). MLVA-genotype analysis of the L. pneumophila isolates and their spatial distribution indicated three niches characterized by distinct physico-chemical parameters and inhabited by specific consortia of genotypes. This study provides novel insights into mechanisms shaping L. pneumophila populations and triggering their abundance leading to an understanding of their genotype-specific niches and ecology in support of improved prevention measures.
    • Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections.

      Schiefer, Andrea; Hübner, Marc P; Krome, Anna; Lämmer, Christine; Ehrens, Alexandra; Aden, Tilman; Koschel, Marianne; Neufeld, Helene; Chaverra-Muñoz, Lillibeth; Jansen, Rolf; et al. (PLOS, 2020-12-07)
      Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal-adult-worm killing-treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4-5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.
    • DNA methylation and body mass index from birth to adolescence: meta-analyses of epigenome-wide association studies.

      Vehmeijer, Florianne O L; Küpers, Leanne K; Sharp, Gemma C; Salas, Lucas A; Lent, Samantha; Jima, Dereje D; Tindula, Gwen; Reese, Sarah; Qi, Cancan; Gruzieva, Olena; et al. (BMC, 2020-11-25)
      DNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance, P < 1.06 × 10-7, with a 0.96 standard deviation score (SDS) (standard error (SE) 0.17), 0.32 SDS (SE 0.06), and 0.32 BMI SDS (SE 0.06) higher BMI per 10% increase in methylation, respectively. DNA methylation at nine additional CpGs in the cross-sectional childhood model was associated with BMI at false discovery rate significance. The strength of the associations of DNA methylation at the 187 CpGs previously identified to be associated with adult BMI, increased with advancing age across childhood and adolescence in our analyses. In addition, correlation coefficients between effect estimates for those CpGs in adults and in children and adolescents also increased. Among the top findings for each age range, we observed increasing enrichment for the CpGs that were previously identified in adults (birth Penrichment = 1; childhood Penrichment = 2.00 × 10-4; adolescence Penrichment = 2.10 × 10-7).
    • Multi-omics examination of Q fever fatigue syndrome identifies similarities with chronic fatigue syndrome.

      Raijmakers, Ruud P H; Roerink, Megan E; Jansen, Anne F M; Keijmel, Stephan P; Gacesa, Ranko; Li, Yang; Joosten, Leo A B; van der Meer, Jos W M; Netea, Mihai G; Bleeker-Rovers, Chantal P; et al. (BMC, 2020-11-26)
      Inflammatory markers, including 4E-BP1 (P = 9.60-16 and 1.41-7) and MMP-1 (P = 7.09-9 and 3.51-9), are significantly more expressed in both QFS and CFS patients compared to HC. Blood metabolite profiles show significant differences when comparing QFS (319 metabolites) and CFS (441 metabolites) patients to HC, and are significantly enriched in pathways like sphingolipid (P = 0.0256 and 0.0033) metabolism. When comparing QFS to CFS patients, almost no significant differences in metabolome were found. Comparison of microbiome taxonomy of QFS and CFS patients with that of HC, shows both in- and decreases in abundancies in Bacteroidetes (with emphasis on Bacteroides and Alistiples spp.), and Firmicutes and Actinobacteria (with emphasis on Ruminococcus and Bifidobacterium spp.). When we compare QFS patients to CFS patients, there is a striking resemblance and hardly any significant differences in microbiome taxonomy are found.
    • Solubility and Stability Enhanced Oral Formulations for the Anti-Infective Corallopyronin A.

      Krome, Anna K; Becker, Tim; Kehraus, Stefan; Schiefer, Andrea; Steinebach, Christian; Aden, Tilman; Frohberger, Stefan J; López Mármol, Álvaro; Kapote, Dnyaneshwar; Jansen, Rolf; et al. (MDPI, 2020-11-18)
      Novel-antibiotics are urgently needed to combat an increase in morbidity and mortality due to resistant bacteria. The preclinical candidate corallopyronin A (CorA) is a potent antibiotic against Gram-positive and some Gram-negative pathogens for which a solid oral formulation was needed for further preclinical testing of the active pharmaceutical ingredient (API). The neat API CorA is poorly water-soluble and instable at room temperature, both crucial characteristics to be addressed and overcome for use as an oral antibiotic. Therefore, amorphous solid dispersion (ASD) was chosen as formulation principle. The formulations were prepared by spray-drying, comprising the water-soluble polymers povidone and copovidone. Stability (high-performance liquid chromatography, Fourier-transform-infrared spectroscopy, differential scanning calorimetry), dissolution (biphasic dissolution), and solubility (biphasic dissolution, Pion's T3 apparatus) properties were analyzed. Pharmacokinetic evaluations after intravenous and oral administration were conducted in BALB/c mice. The results demonstrated that the ASD formulation principle is a suitable stability- and solubility-enhancing oral formulation strategy for the API CorA to be used in preclinical and clinical trials and as a potential market product.
    • Cholesterol sensing by CD81 is important for hepatitis C virus entry.

      Palor, Machaela; Stejskal, Lenka; Mandal, Piya; Lenman, Annasara; Alberione, Maria Pia; Kirui, Jared; Moeller, Rebecca; Ebner, Stefan; Meissner, Felix; Gerold, Gisa; et al. (DeGruyter, 2020-09-08)
      CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease.
    • MicroRNA-342-3p is a potent tumour suppressor in hepatocellular carcinoma.

      Komoll, Ronja-Melinda; Hu, Qingluan; Olarewaju, Olaniyi; von Döhlen, Lena; Yuan, Qinggong; Xie, Yu; Tsay, Hsin-Chieh; Daon, Joel; Qin, Renyi; Manns, Michael P; et al. (Elsevier, 2020-07-30)
      Background & aims: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. Methods: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. Results: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. Conclusions: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. Lay summary: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.
    • Impact of process temperature and organic loading rate on cellulolytic/hydrolytic biofilm microbiomes during biomethanation of ryegrass silage revealed by genome-centered metagenomics and metatranscriptomics

      Maus, Irena; Klocke, Michael; Derenkó, Jaqueline; Stolze, Yvonne; Beckstette, Michael; Jost, Carsten; Wibberg, Daniel; Blom, Jochen; Henke, Christian; Willenbücher, Katharina; et al. (BMC, 2020-03-02)
      Background: Anaerobic digestion (AD) of protein-rich grass silage was performed in experimental two-stage twophase biogas reactor systems at low vs. increased organic loading rates (OLRs) under mesophilic (37 °C) and thermophilic (55 °C) temperatures. To follow the adaptive response of the biomass-attached cellulolytic/hydrolytic biofilms at increasing ammonium/ammonia contents, genome-centered metagenomics and transcriptional profiling based on metagenome assembled genomes (MAGs) were conducted. Results: In total, 78 bacterial and archaeal MAGs representing the most abundant members of the communities, and featuring defined quality criteria were selected and characterized in detail. Determination of MAG abundances under the tested conditions by mapping of the obtained metagenome sequence reads to the MAGs revealed that MAG abundance profiles were mainly shaped by the temperature but also by the OLR. However, the OLR effect was more pronounced for the mesophilic systems as compared to the thermophilic ones. In contrast, metatranscriptome mapping to MAGs subsequently normalized to MAG abundances showed that under thermophilic conditions, MAGs respond to increased OLRs by shifting their transcriptional activities mainly without adjusting their proliferation rates. This is a clear difference compared to the behavior of the microbiome under mesophilic conditions. Here, the response to increased OLRs involved adjusting of proliferation rates and corresponding transcriptional activities. The analysis led to the identification of MAGs positively responding to increased OLRs. The most outstanding MAGs in this regard, obviously well adapted to higher OLRs and/or associated conditions, were assigned to the order Clostridiales (Acetivibrio sp.) for the mesophilic biofilm and the orders Bacteroidales (Prevotella sp. and an unknown species), Lachnospirales (Herbinix sp. and Kineothrix sp.) and Clostridiales (Clostridium sp.) for the thermophilic biofilm. Genome-based metabolic reconstruction and transcriptional profiling revealed that positively responding MAGs mainly are involved in hydrolysis of grass silage, acidogenesis and / or acetogenesis. Conclusions: An integrated -omics approach enabled the identification of new AD biofilm keystone species featuring outstanding performance under stress conditions such as increased OLRs. Genome-based knowledge on the metabolic potential and transcriptional activity of responsive microbiome members will contribute to the development of improved microbiological AD management strategies for biomethanation of renewable biomass. Keywords: Metagenome assembled genomes, Integrated -omics, Polyomics, Anaerobic digestion, Biogas, Bioconversion, Microbial community structure, Methane, Metabolic activity
    • MicroRNA-342-3p is a potent tumour suppressor in hepatocellular carcinoma

      Komoll, Ronja Melinda; Hu, Qingluan; Olarewaju, Olaniyi; von Döhlen, Lena; Yuan, Qinggong; Xie, Yu; Tsay, Hsin Chieh; Daon, Joel; Qin, Renyi; Manns, Michael P.; et al. (2021-01-01)
      Background & aims: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. Methods: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. Results: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. Conclusions: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. Lay summary: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.
    • Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections.

      Gödecke, Natascha; Riedel, Jan; Herrmann, Sabrina; Behme, Sara; Rand, Ulfert; Kubsch, Tobias; Cicin-Sain, Luka; Hauser, Hansjörg; Köster, Mario; Wirth, Dagmar; et al. (Oxford Academic, 2020-11-18)
      Mammalian first line of defense against viruses is accomplished by the interferon (IFN) system. Viruses have evolved numerous mechanisms to reduce the IFN action allowing them to invade the host and/or to establish latency. We generated an IFN responsive intracellular hub by integrating the synthetic transactivator tTA into the chromosomal Mx2 locus for IFN-based activation of tTA dependent expression modules. The additional implementation of a synthetic amplifier module with positive feedback even allowed for monitoring and reacting to infections of viruses that can antagonize the IFN system. Low and transient IFN amounts are sufficient to trigger these amplifier cells. This gives rise to higher and sustained-but optionally de-activatable-expression even when the initial stimulus has faded out. Amplification of the IFN response induced by IFN suppressing viruses is sufficient to protect cells from infection. Together, this interfaced sensor/actuator system provides a toolbox for robust sensing and counteracting viral infections.
    • Unsaturated Fatty Acids Control Biofilm Formation of and Other Gram-Positive Bacteria.

      Yuyama, Kamila Tomoko; Rohde, Manfred; Molinari, Gabriella; Stadler, Marc; Abraham, Wolf-Rainer; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-11-08)
      Infections involving biofilms are difficult to treat due to increased resistances against antibiotics and the immune system. Hence, there is an urgent demand for novel drugs against biofilm infections. During our search for novel biofilm inhibitors from fungi, we isolated linoleic acid from the ascomycete Hypoxylon fragiforme which showed biofilm inhibition of several bacteria at sub-MIC concentrations. Many fatty acids possess antimicrobial activities, but their minimum inhibitory concentrations (MIC) are high and reports on biofilm interferences are scarce. We demonstrated that not only linoleic acid but several unsaturated long-chain fatty acids inhibited biofilms at sub-MIC concentrations. The antibiofilm activity exerted by long-chain fatty acids was mainly against Gram-positive bacteria, especially against Staphylococcus aureus. Micrographs of treated S. aureus biofilms revealed a reduction in the extracellular polymeric substances, pointing to a possible mode of action of fatty acids on S. aureus biofilms. The fatty acids had a strong species specificity. Poly-unsaturated fatty acids had higher activities than saturated ones, but no obvious rule could be found for the optimal length and desaturation for maximal activity. As free fatty acids are non-toxic and ubiquitous in food, they may offer a novel tool, especially in combination with antibiotics, for the control of biofilm infections.
    • MicroRNA-221: A Fine Tuner and Potential Biomarker of Chronic Liver Injury.

      Markovic, Jovana; Sharma, Amar Deep; Balakrishnan, Asha; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-07-23)
      The last decade has witnessed significant advancements in our understanding of how small noncoding RNAs, such as microRNAs (miRNAs), regulate disease progression. One such miRNA, miR-221, has been shown to play a key role in the progression of liver fibrosis, a common feature of most liver diseases. Many reports have demonstrated the upregulation of miR-221 in liver fibrosis caused by multiple etiologies such as viral infections and nonalcoholic steatohepatitis. Inhibition of miR-221 via different strategies has shown promising results in terms of the suppression of fibrogenic gene signatures in vitro, as well as in vivo, in independent mouse models of liver fibrosis. In addition, miR-221 has also been suggested as a noninvasive serum biomarker for liver fibrosis and cirrhosis. In this review, we discuss the biology of miR-221, its significance and use as a biomarker during progression of liver fibrosis, and finally, potential and robust approaches that can be utilized to suppress liver fibrosis via inhibition of miR-221.
    • Notch and TLR signaling coordinate monocyte cell fate and inflammation.

      Gamrekelashvili, Jaba; Kapanadze, Tamar; Sablotny, Stefan; Ratiu, Corina; Dastagir, Khaled; Lochner, Matthias; Karbach, Susanne; Wenzel, Philip; Sitnow, Andre; Fleig, Susanne; et al. (elife Sciences, 2020-07-29)
      Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.
    • Epigenome-wide association study of DNA methylation and adult asthma in the Agricultural Lung Health Study.

      Hoang, Thanh T; Sikdar, Sinjini; Xu, Cheng-Jian; Lee, Mi Kyeong; Cardwell, Jonathan; Forno, Erick; Imboden, Medea; Jeong, Ayoung; Madore, Anne-Marie; Qi, Cancan; et al. (European Respiratory Society (ERS), 2020-09-03)
      Epigenome-wide studies of methylation in children support a role for epigenetic mechanisms in asthma; however, studies in adults are rare and few have examined non-atopic asthma. We conducted the largest epigenome-wide association study (EWAS) of blood DNA methylation in adults in relation to non-atopic and atopic asthma.We measured DNA methylation in blood using the Illumina MethylationEPIC array among 2286 participants in a case-control study of current adult asthma nested within a United States agricultural cohort. Atopy was defined by serum specific immunoglobulin E (IgE). Participants were categorised as atopy without asthma (n=185), non-atopic asthma (n=673), atopic asthma (n=271), or a reference group of neither atopy nor asthma (n=1157). Analyses were conducted using logistic regression.No associations were observed with atopy without asthma. Numerous cytosine-phosphate-guanine (CpG) sites were differentially methylated in non-atopic asthma (eight at family-wise error rate (FWER) p<9×10-8, 524 at false discovery rate (FDR) less than 0.05) and implicated 382 novel genes. More CpG sites were identified in atopic asthma (181 at FWER, 1086 at FDR) and implicated 569 novel genes. 104 FDR CpG sites overlapped. 35% of CpG sites in non-atopic asthma and 91% in atopic asthma replicated in studies of whole blood, eosinophils, airway epithelium, or nasal epithelium. Implicated genes were enriched in pathways related to the nervous system or inflammation.We identified numerous, distinct differentially methylated CpG sites in non-atopic and atopic asthma. Many CpG sites from blood replicated in asthma-relevant tissues. These circulating biomarkers reflect risk and sequelae of disease, as well as implicate novel genes associated with non-atopic and atopic asthma.
    • Characterization of Populations by Multilocus Variable Number of Tandem Repeats (MLVA) Genotyping from Drinking Water and Biofilm in Hospitals from Different Regions of the West Bank.

      Zayed, Ashraf R; Pecellin, Marina; Salah, Alaa; Alalam, Hanna; Butmeh, Suha; Steinert, Michael; Lesnik, Rene; Brettar, Ingrid; Höfle, Manfred G; Bitar, Dina M; et al. (MDPI, 2020-10-22)
      The West Bank can be considered a high-risk area for Legionnaires' disease (LD) due to its hot climate, intermittent water supply and roof storage of drinking water. Legionella, mostly L. pneumophila, are responsible for LD, a severe, community-acquired and nosocomial pneumonia. To date, no extensive assessment of Legionella spp and L. pneumophila using cultivation in combination with molecular approaches in the West Bank has been published. Two years of environmental surveillance of Legionella in water and biofilms in the drinking water distribution systems (DWDS) of eight hospitals was carried out; 180 L. pneumophila strains were isolated, mostly from biofilms in DWDS. Most of the isolates were identified as serogroup (Sg) 1 (60%) and 6 (30%), while a minor fraction comprised Sg 8 and 10. Multilocus Variable number of tandem repeats Analysis using 13 loci (MLVA-8(12)) was applied as a high-resolution genotyping method and compared to the standard Sequence Based Typing (SBT). The isolates were genotyped in 27 MLVA-8(12) genotypes (Gt), comprising four MLVA clonal complexes (VACC 1; 2; 5; 11). The major fraction of isolates constituted Sequence Type (ST)1 and ST461. Most of the MLVA-genotypes were highly diverse and often unique. The MLVA-genotype composition showed substantial regional variability. In general, the applied MLVA-method made it possible to reproducibly genotype the isolates, and was consistent with SBT but showed a higher resolution. The advantage of the higher resolution was most evident for the subdivision of the large strain sets of ST1 and ST461; these STs were shown to be highly pneumonia-relevant in a former study. This shows that the resolution by MLVA is advantageous for back-tracking risk sites and for the avoidance of outbreaks of L. pneumophila. Overall, our results provide important insights into the detailed population structure of L. pneumophila, allowing for better risk assessment for DWDS.
    • RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression.

      Knittel, Vanessa; Sadana, Pooja; Seekircher, Stephanie; Stolle, Anne-Sophie; Körner, Britta; Volk, Marcel; Jeffries, Cy M; Svergun, Dmitri I; Heroven, Ann Kathrin; Scrima, Andrea; et al. (PLOS, 2020-09-23)
      Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.
    • Impact of Von Willebrand Factor on Bacterial Pathogenesis.

      Steinert, Michael; Ramming, Isabell; Bergmann, Simone; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2020-09-03)
      Von Willebrand factor (VWF) is a mechano-sensitive protein with crucial functions in normal hemostasis, which are strongly dependant on the shear-stress mediated defolding and multimerization of VWF in the blood stream. Apart from bleeding disorders, higher plasma levels of VWF are often associated with a higher risk of cardiovascular diseases. Herein, the disease symptoms are attributed to the inflammatory response of the activated endothelium and share high similarities to the reaction of the host vasculature to systemic infections caused by pathogenic bacteria such as Staphylococcus aureus and Streptococcus pneumoniae. The bacteria recruit circulating VWF, and by binding to immobilized VWF on activated endothelial cells in blood flow, they interfere with the physiological functions of VWF, including platelet recruitment and coagulation. Several bacterial VWF binding proteins have been identified and further characterized by biochemical analyses. Moreover, the development of a combination of sophisticated cell culture systems simulating shear stress levels of the blood flow with microscopic visualization also provided valuable insights into the interaction mechanism between bacteria and VWF-strings. In vivo studies using mouse models of bacterial infection and zebrafish larvae provided evidence that the interaction between bacteria and VWF promotes bacterial attachment, coagulation, and thrombus formation, and thereby contributes to the pathophysiology of severe infectious diseases such as infective endocarditis and bacterial sepsis. This mini-review summarizes the current knowledge of the interaction between bacteria and the mechano-responsive VWF, and corresponding pathophysiological disease symptoms.
    • Recombinant protein production-associated metabolic burden reflects anabolic constraints and reveals similarities to a carbon overfeeding response.

      Li, Zhaopeng; Rinas, Ursula; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-09-03)
      A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.
    • Strategic Anti-SARS-CoV-2 Serology Testing in a Low Prevalence Setting: The COVID-19 Contact (CoCo) Study in Healthcare Professionals.

      Behrens, Georg M N; Cossmann, Anne; Stankov, Metodi V; Schulte, Bianca; Streeck, Hendrik; Förster, Reinhold; Bosnjak, Berislav; Willenzon, Stefanie; Boeck, Anna-Lena; Thu Tran, Anh; et al. (Springer Healthcare, 2020-09-04)
      Background: Serology testing is explored for epidemiological research and to inform individuals after suspected infection. During the coronavirus disease 2019 (COVID-19) pandemic, frontline healthcare professionals (HCP) may be at particular risk for infection. No longitudinal data on functional seroconversion in HCP in regions with low COVID-19 prevalence and low pre-test probability exist. Methods: In a large German university hospital, we performed weekly questionnaire assessments and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) measurements with various commercial tests, a novel surrogate virus neutralisation test, and a neutralisation assay using live SARS-CoV-2. Results: From baseline to week 6, 1080 screening measurements for anti-SARS CoV-2 (S1) IgG from 217 frontline HCP (65% female) were performed. Overall, 75.6% of HCP reported at least one symptom of respiratory infection. Self-perceived infection probability declined over time (from mean 20.1% at baseline to 12.4% in week 6, p < 0.001). In sera of convalescent patients with PCR-confirmed COVID-19, we measured high anti-SARS-CoV-2 IgG levels, obtained highly concordant results from enzyme-linked immunosorbent assays (ELISA) using e.g. the spike 1 (S1) protein domain and the nucleocapsid protein (NCP) as targets, and confirmed antiviral neutralisation. However, in HCP the cumulative incidence for anti-SARS-CoV-2 (S1) IgG was 1.86% for positive and 0.93% for equivocal positive results over the study period of 6 weeks. Except for one HCP, none of the eight initial positive results were confirmed by alternative serology tests or showed in vitro neutralisation against live SARS-CoV-2. The only true seroconversion occurred without symptoms and mounted strong functional humoral immunity. Thus, the confirmed cumulative incidence for neutralizing anti-SARS-CoV-2 IgG was 0.47%. Conclusion: When assessing anti-SARS-CoV-2 immune status in individuals with low pre-test probability, we suggest confirming positive results from single measurements by alternative serology tests or functional assays. Our data highlight the need for a methodical serology screening approach in regions with low SARS-CoV-2 infection rates.